This invention is concerned with recombinant DNA molecules, and methods for producing proteins by said molecules. The invention is further concerned with recombinant DNA molecules that are synthesized in Bacillus strain bacteria and are known to have DNA which codes for exoenzymes excreted by them and that are present in tens of copies of Bacillus strain bacteria; as well as with recombinant DNA molecules modified from the above recombinant DNA molecules that are known to have DNA which contains the regulation and excretion signals of the .alpha.-amylase gene of B. amyloliquefaciens, to which signals a gene of any protein can be joined. The invention is particularly related to the regulation and deleted, non-functional excretion signal of the .alpha.-amylase gene of B. amyloliquefaciens, to which a desired structural protein may be joined. It has been found that, for certain structural proteins, Bacillus expression may be surprisingly enhanced, and is to be desired, when intracellular production is achieved according to this aspect of the invention. As will be described in the following, these recombinant DNA molecules can be used, for example, to intensify the .alpha.-amylase production in Bacillus strain bacteria, and their modifications to produce any desired homologous or heterologous protein or peptide in Bacillus strain bacteria.
Recent development in molecular biology has created new possibilities for protein production in bacteria by recombinant DNA techniques. In addition to the possibility of producing proteins of eukaryotic cells in bacteria by recombinant DNA techniques, the synthesis of the proteins of the bacteria themselves can be significantly improved by increasing the number of the copies of the desired gene in the cell. The number of the gene copies in a bacterium cell can be increased by joining the gene to such a plasmid or virus DNA molecule as is found in the cell in several, usually 10 to 100, copies. The increased number of the gene copies in a cell usually also leads to a corresponding increase in the protein synthesis expressed by the gene.
Even though several experiments of this type have been carried out using E. coli and plasmid or virus DNA molecules replicating in it as host bacterium, the use of Bacillus strain bacteria as hosts is only beginning (Gryczan et al., Molecular General Genet. 117:459-467 (1979); Keggins et al., Proc. Natl. Acad. Sci. USA 75:1423-1427 (1978); Yoneda et al., Biochem. Biophys. Res. Commun. 91:1556-1564 (1979)).